Stable dry powder composition comprising biologically active microorganisms and/or bioactive materials and methods of making

ABSTRACT

The present invention relates to embedding live or dead microorganisms and/or bioactive materials in a protective dry formulation matrix, wherein the formulation includes the bioactive microorganism or material, a formulation stabilizer agent, and a protective agent. The formulation is prepared by dispersing all the solid components in a solution, with or without a vacuum, and cooling the solution to a temperature above its freezing temperature. The methods include a primary drying step of the formulation at a desired temperature and time period, and an accelerated secondary drying step under maximum vacuum and elevated temperature, to achieve a final desirable water activity of the dry material.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.16/227,075, filed Dec. 20, 2018, which is a continuation of U.S.application Ser. No. 13/321,708, filed Feb. 6, 2012, which is a U.S.National Phase Application of PCT International ApplicationPCT/US2010/036098, filed May 26, 2010, which claims priority to U.S.Provisional Application Nos. 61/181,248 and 61/223,295 filed May 26,2009 and Jul. 6, 2009, respectively, the contents of each of which arehereby incorporated by reference herein for all purposes.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is in the field of protection of bioactivemicroorganism and/or materials in high temperature and humid conditions.In particular, the invention relates to embedding live microorganismsand/or bioactive materials in a protective dry formulation matrix.

Related Background Art

Bioactive microorganisms, such as live or dead bacteria and viruses, orbioactive materials, such as proteins, vitamins, minerals, hormones andcells are generally unstable when stored under conditions of hightemperature and humidity. For example, many commercially availableprobiotic bacteria such as Lactobacillus rhamnosus can loose more thanone log of viability in less than two weeks when stored in ambientatmosphere at room temperature (approximately 25° C.). A common processto dry and protect these bioactive microorganisms after harvesting froma culture vessel (e.g., fermentor) is to drop a concentrated solution ofthe living cells into liquid nitrogen then store the frozen beads in−80° C. refrigeration for later freeze drying or shipment to otherlocations. Freeze-drying has been a dominant method for drying sensitivebioactive material. Other methods, such as spray drying, 5464332_1supercritical fluid drying, and desiccation are generally not suitablefor sensitive bioactives such as live or attenuated bacteria and virusesbecause of the high drying temperatures used in these methods whichresult in significant damage to the microorganism itself. In addition,they may not sufficiently dry the material to the specific residualmoisture requirements for product stability, and thus an additionaldrying step by other means, may be required.

In freeze-drying, the bioactive microorganism or materials is commonlymixed in a solution or suspension of protective agents, frozen, and thendehydrated by sublimation under full vacuum. The low temperatures of thefreeze-drying process decrease the degradation reactions of thebioactive and minimize the loss of activity in the final dry form.However, the requirement for sub-zero temperatures is energy intensive,and the low surface area to volume ratios of the frozen materialnecessitates the use of long drying time (up to several days per batchcycle). The slow drying of the freeze-drying process also facilitatesthe formation of ice crystals that can damage or denature a sensitivebioactive. For this reason, bioactive microorganism or materials such asviruses, bacteria, and cells that possess a cell wall or lipid membrane,pose significant challenges to the freeze-drying process.

One option to reduce the formation of an ice crystal structure is to addcryoprotective agents to the bioactive solution. Such protective agentsare highly soluble chemicals that are added to a formulation to protectcell membranes and intracellular proteins during freezing and to enhancestability during storage. Common stabilizers for live bacteria andviruses include sugars such as sucrose, glycerol, or sorbitol, at highconcentrations with the cellular material or bioactive (Morgan et al.,2006; Capela et al., 2006). However, such protective agents may notpenetrate adequately into the cell to protect active components withinthe intracellular volume. Therefore, a significant challenge remains todevelop an optimal drying process and formulation that minimizes dryinglosses while achieving adequate storage stability of the dried material.

Some of the problems associated with the freeze-drying have beenresolved by using a combination of certain formulations and vacuumdrying in a liquid state. Annear (Annear 1962) developed a formulationcontaining bacteria in a solution of sugars and amino acids and a vacuumdrying process that involves boiling and foam formation. Roser et al.(U.S. Pat. No. 6,964,771) disclosed a similar concept of drying by foamformation that includes a liquid concentration step followed by boilingand foaming the concentrated solution (syrup) under vacuum. To mitigatethe oxidation and denaturation damage that can occur during the boilingstep, Bronshtein (U.S. Pat. Nos. 5,766,520, 7,153,472) introduced animproved protective formula containing carbohydrates and surfactants.The drying of the protective solution also involved a stepwise processof concentration under a moderate vacuum before application of a strongvacuum to cause frothy boiling of the remaining water to form dry stablefoam. In an attempt to eliminate the boiling step, Busson and Schroeder(U.S. Pat. No. 6,534,087) have proposed a drying process in a liquidstate formulation for insensitive bioactives using a vacuum oven withoutboiling, by applying very mild vacuum pressure above 30 Torr. Afterachieving a certain level of drying without boiling the material, heatwas applied at above 20° C. and dried material was harvested after onlya few hours.

This type of drying process, in which the bioactive solution ismaintained in a liquid state during the entire drying process, has theadvantage of faster drying due to convection of the liquid duringboiling and the increased surface area presented by the foamingsurfaces. However, boiling and foaming require input of a significantamount of heat to provide the necessary eruption of the solution. Such adrying process is not well adapted to drying of sensitive biologicals,such as viable viruses, cells or bacteria because the applied heataccelerates enzymatic processes (e.g., proteolysis), and chemicalprocesses (e.g., oxidation and free radical attacks), which can destroythe activity or viability of the biological material.

The drying process described above is also limited in its ability to bescaled to a large industrial process. The avoidance of freezing requiresthe process to be conducted at lower vacuum level (>7 Torr) than inconventional freeze drying or spray freeze drying process cycles. Themost significant disadvantage of the above processes is the inability tocontrol and limit the expansion of the foam within the vessel, tray orvial. The uncontrollable eruption and often-excessive foam formationmakes it practically impossible to develop an industrial scale process.The eruption and foaming nature of the boiling step results in a portionof material being splattered on the walls of the vessel and into thedrying chamber. To soften the eruption during boiling, Bronshtein (U.S.Pat. Nos. 6,884,866, 6,306,345) has proposed special chambers and acontrolled temperature/pressure application protocol that reducesoverheating to an acceptable level. Another approach to contain theeruption and excessive foaming is described in US. Pat. App. No.:2008/0229609, in which the bioactive solution is enclosed in a containeror a bag covered with breathable membranes. Once again, these protocolsare difficult to implement in industrial level and they are difficult toreliably replicate with different formulations.

A need remains for a suitable protective formulation that can be driedin a liquid state and an industrially scaleable method to dry bioactivemicroorganisms such as live or dead viruses, bacteria and cells,particularly at temperatures above freezing. There is a needparticularly for a cost effective scaleable drying process that is alsosuitable for applications outside the pharmaceutical industry such asfood and agriculture industries. Protective formulations and mild dryingprocesses are required to provide adequate drying without exposure tohigh temperatures. A composition is needed that can protect suchbiologicals in storage under high temperature and humid conditions. Thepresent invention, as described below, provides a solution to all ofthese challenges.

SUMMARY OF THE INVENTION

The present invention includes compositions and methods for preservingbioactive materials, such as peptides, proteins, hormones, vitamins,minerals, drugs, microbiocides, fungicides, herbicides, insecticides,spermicides, nucleic acids, antibodies, vaccines, and/or bioactivemicroorganism such as bacteria (probiotic or otherwise), viruses and/orcell suspensions, in storage. The drying methods provide a process ofcontrollable expansion of a formulation comprising the bioactivemicroorganism or material, a formulation stabilizer agent, and aprotective agent. The formulation is prepared by dispersing all thesolid components in a solution, with or without a vacuum. The solutionis cooled to a temperature above its freezing temperature and driedunder vacuum into a dry composition, which exhibits an unexpectedly highstability. The methods include a primary drying step of the formulationat a desired temperature and time period, and an accelerated secondarydrying step under maximum vacuum and elevated temperature, to achieve afinal desirable water activity of the dry material.

In one embodiment, the formulation comprises sufficient amounts offormulation stabilizer agents, in which the microorganisms are embedded.Examples of a suitable formulation stabilizer agent include, but are notlimited to, cellulose acetate phthalate (CAP), carboxy-methyl-cellulose,pectin, sodium alginate, salts of alginic acid, hydroxyl propyl methylcellulose (HPMC), methyl cellulose, carrageenan, guar gum, gum acacia,xanthan gum, locust bean gum, chitosan and chitosan derivatives,collagen, polyglycolic acid, starches and modified starches,cyclodextrins and oligosaccharides (inulin, maltodextrins, dextrans,etc.); and combinations thereof.

In one particular embodiment, the preferred formulation stabilizer agentis sodium alginate. Preferably, the formulation comprises, in percent byweight of total dry matter, 0.1-10%, preferably 1-6%, more preferably2-4% of formulation stabilizer agent. In an additional embodiment, theformulation stabilizer comprises a mixture of sodium alginate andoligosaccharides in a weight ratio of 1:1-10, more preferably 1:1-5 ofsodium alginate/oligosaccharides. In yet another embodiment of thepresent invention, the formulation stabilizer is cross-linked withdivalent metals ions to form a firm hydrogel.

In another embodiment, the formulation comprises significant amounts ofprotecting agents, in which the microorganisms are embedded. Examples ofa suitable protecting agent include but not limited to proteins such ashuman and bovine serum albumin, egg albumen, gelatin, immunoglobulin,isolated soya protein, wheat protein, skim milk powder, caseinate, wheyprotein and any protein hydrolysates; carbohydrates includingmonosaccharides (e.g., galactose, D-mannose, sorbose, etc.),disaccharides (e.g., lactose, trehalose, sucrose, etc.), an amino acidsuch as lysine, monosodium glutamate, glycine, alanine, arginine orhistidine, as well as hydrophobic amino acids (tryptophan, tyrosine,leucine, phenylalanine, etc.); a methylamine such as betaine; anexcipient salt such as magnesium sulfate; a polyol such as trihydric orhigher sugar alcohols, (e.g. glycerin, erythritol, glycerol, arabitol,xylitol, sorbitol, and mannitol); propylene glycol; polyethylene glycol;pluronics; surfactants; and combinations thereof.

In one preferred embodiment, the protecting agent comprises a mixture ofa disaccharide, a protein, and a protein hydrolysate. In a particularembodiment, the preferred protecting agent is a mixture of trehalose,soy protein isolate or whey protein and their hydrolysates. Preferably,the formulation comprises, in percent by weight of total dry matter,10-90%, of trehalose, 0.1-30% soy protein isolate or whey proteins and0.1-30% soy or whey protein hydrolysate. Preferably 20-80% of trehalose,0.1-20% soy protein isolate or whey proteins and 1-20% soy or wheyprotein hydrolysate, more preferably 40-80% of trehalose, 0.1-20% soyprotein isolate or whey proteins and 1-20% soy or whey proteinhydrolysate.

The method of the invention typically includes blending with or withouta vacuum, concentrated solution or dry powder of bioactive microorganism(e.g., live or dead vaccines, bacteria, algae, viruses and/or cellsuspensions) or a bioactive material (e.g., peptides, proteins,hormones, vitamins, minerals, drugs, microbiocides, fungicides,herbicides, insecticides, spermicides, nucleic acids, antibodies,vaccines), a stabilizer agent, and a protective agent into a homogeneousformulation, cooling the formulation to a temperature above its freezingtemperature, and drying under vacuum at a shelf temperature above 20° C.According to the invention, the drying process can involve a primaryvacuum drying at a shelf temperature of 20° C. or above, followed by anaccelerating secondary drying of the formulation under maximum vacuumand elevated temperature for a time sufficient to reduce the wateractivity of the dried formulation to 0.3 Aw or less.

In one embodiment of the mixing method the bioactive microorganism ormaterial is in a dry stabilized form and is further dry blended with thedry stabilizer agents and protective agents. This dry blend is thenadded to water and mixed under the appropriate vacuum and agitation togive a homogeneous slurry of the desired density.

In another embodiment of the mixing method, the bioactive microorganismor material is in the form of a concentrated solution or paste. Thesolution is mixed with all the other formulation ingredients beforeadding to water.

In yet another embodiment of the mixing method, the bioactivemicroorganism or material is in the form of dry powder. The dry powderis mixed with all the other formulation ingredients before adding towater.

In another embodiment of the mixing method, the dry bioactivemicroorganism or material is mixed with just a portion of theformulation ingredients, and this mixture is added to the pre-formedslurry, prepared from the addition of the other formulation ingredientsto water.

In preferred embodiments of the drying methods, the bioactivemicroorganism is mixed under vacuum in a solution including aformulation stabilizer agent and a protective agent. In one particularembodiment, the bioactive microorganism comprises live bacteria (e.g.,probiotic bacteria). Examples of suitable microorganisms include, butare not limited to, yeasts such as Saccharomyces, Debaromyces, Candida,Pichia and Torulopsis, moulds such as Aspergillus, Rhizopus, Mucor,Penicillium and Torulopsis and bacteria such as the generaBifidobacterium, Clostridium, Fusobacterium, Melissococcus,Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Kocuriaw,Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus,Leuconostoc, Weissella, Aerococcus, Oenococcus and Lactobacillus.Specific examples of suitable probiotic microorganisms would berepresented by the following species and include all culture biotypeswithin those species: Aspergillus niger, A. oryzae, Bacillus coagulans,B. lentus, B. licheniformis, B. mesentericus, B. pumilus, B. subtilis,B. natto, Bacteroides amylophilus, Bac. capillosus, Bac. ruminocola,Bac. suis, Bifidobacterium adolescentis, B. animalis, B. breve, B.bifidum, B. infantis, B. lactis, B. longum, B. pseudolongum, B.thermophilum, Candida pintolepesii, Clostridium butyricum, Enterococcuscremoris, E. diacetylactis, E faecium, E. intermedius, E. lactis, E.muntdi, E. thermophilus, Escherichia coli, Kluyveromyces fragilis,Lactobacillus acidophilus, L. alimentarius, L. amylovorus, L. crispatus,L. brevis, L. case 4 L. curvatus, L. cellobiosus, L. delbrueckii ss.bulgaricus, L. farciminis, L. fermentum, L. gasseri, L. helveticus, L.lactis, L. plantarum, L. johnsonii, L. reuteri, L. rhamnosus, L. sakei,L. salivarius, Leuconostoc mesenteroides, P. cereviseae (damnosus),Pediococcus acidilactici, P. pentosaceus, Propionibacteriumfreudenreichii, Prop. shermanii, Saccharomyces cereviseae,Staphylococcus carnosus, Staph. xylosus, Streptococcus infantarius,Strep. salivarius ss. thermophilus, Strep. Thermophilus and Strep.lactis.

In preferred methods, the formulation is mixed under vacuum at roomtemperature (e.g., from 20° C. to 30° C.). After mixing to homogeneity,the formulation is then cooled to a temperature above the freezingtemperature of the formulation. Typically, the formulation is cooled tobetween −10° C. to +10° C., more preferably the formulation is cooled tobetween −5° C. and +5° C. In a preferred embodiment, the cooledformulation is then transferred to a drying chamber where heating isapplied (20° C. or more) while controlling an initial vacuum pressure ata level to maintain the original pre-cooling temperature. Typically, thedesirable vacuum pressure is below 7 Torr but no less than 3 Torr. Underthese preferred conditions a controlled expansion of the formulation andsubsequent faster primary drying of the formulation is achieved. Toaccelerate the secondary drying, a maximum vacuum pressure is appliedand heat supply temperature may be further elevated to from 30° C. to60° C. To maximize the stability of the final product the formulation ispreferably dried for a time sufficient to reduce the water activity ofthe formulation to Aw=0.3 or less. In a preferred embodiment of theinvention, the secondary drying comprises removal of water at a pressureof less than 1 Torr, and in an especially preferred embodiment to lessthan 0.2 Torr.

The wet formulation can be in the form of viscous slurry or hydrogelparticles ranging from 0.05 to 10 mm. The dried formulation can be useddirectly as a flake, or ground into a powder with an average particlesize from about 10 μm to about 1000 μm. The formulation can beadministrated directly to an animal, including human, as a concentratedpowder, as a reconstituted liquid, (e.g., beverage), or it can beincorporated either in flake or powder form into an existing food orfeed product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the stability trend of the probiotic bacteria, L.rhamnosus, which was subjected to storage at 40° C. and 33% relativehumidity.

FIG. 2 shows the process temperatures and cumulative viability loss fora formulation process ending with Aw of 0.28 secondary drying step.

FIG. 3 shows the effect of alginate viscosity on the formulationexpansion under vacuum.

FIG. 4 shows the effect of different formulation stabilizers on storagestability.

FIG. 5 shows the effect of different combinations of stabilizer agentson bacteria viability.

FIG. 6 shows the effect of the formulation density on expansion rateunder vacuum.

FIG. 7 shows the effect of the formulation pre-cooling temperature onexpansion under vacuum.

FIG. 8 shows the effect of the vacuum pressure on formulationtemperature during primary drying step.

FIG. 9 shows the effect of the vacuum pressure on drying rate of theformulation.

FIG. 10 shows the stability of the probiotic bacteria, L. acidophilusdried with the formulation and method of the invention under storage at37° C. and 33% relative humidity.

FIG. 11 shows a flow chart of the method of production stable dryformulation from hydrogel formulation according to the invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a”, “an” and “the” include plural referents unlessthe content clearly dictates otherwise. Thus, for example, reference to“a protein” includes singular protein or a combination of two or moreproteins; reference to “enzyme”, “vitamin”, “bacteria”, etc., includessingular or mixtures of several, and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

“Ambient” room temperatures or conditions are those at any given time ina given environment. Typically, ambient room temperature is 22-25° C.,ambient atmospheric pressure, and ambient humidity are readily measuredand will vary depending on the time of year, weather and climacticconditions, altitude, etc.

“Degassing” refers to the release of a gas from solution in a liquidwhen the partial pressure of the gas is greater than the appliedpressure. This is not boiling, and can often occur at pressures above apressure that would boil a solution. For example, bottled carbonatedsoft drinks contained a high partial pressure of CO₂. Removing thebottle cap reduces the partial pressure and the drink bubbles vigorously(it degasses, but does not boil).

“Boiling” refers to the rapid phase transition from liquid to gas thattakes place when the temperature of a liquid is above its boilingtemperature. The boiling temperature is the temperature at which thevapor pressure of a liquid is equal to the applied pressure. Boiling canbe particularly vigorous when heat is added to a liquid that is alreadyat its boiling point.

“Water activity” or “Aw” in the context of dried formulationcompositions, refers to the availability of water and represents theenergy status of the water in a system. It is defined as the vaporpressure of water above a sample divided by that of pure water at thesame temperature. Pure distilled water has a water activity of exactlyone or Aw=1.0.

“Relative Humidity” or “RH” in the context of storage stability refersto the amount of water vapor in the air at a given temperature. Relativehumidity is usually less than that required to saturate the air andexpressed in percent of saturation humidity.

“Primary drying”, with regard to processes described herein, refers tothe drying that takes place from the time of initial vacuum applicationto the point where secondary drying starts. Typically, the bulk ofprimary drying takes place by extensive evaporation, while the producttemperature remained significantly lower than the temperatures of theheat source.

“Secondary drying”, with regard to processes described herein, refers toa drying step that takes place at temperatures above freezingtemperatures of the formulation and near the temperature of the heatsource. In a typical formulation drying process, a secondary drying stepreduces the water activity of the formulation to an Aw of 0.3 or less.

“Bioactive microorganism,” or “biologically active microorganism orformulation” refers to live or dead microorganism preparations, whichare in such a form as to permit the biological activity of themicroorganism to be unequivocally effective. “Live microorganism as drypowder” refers to a bacterial biomass in which at least 10% W/W is livebacteria. “Dead microorganism as dry powder” refers to a bacterialbiomass in which at least 99.999% is dead bacteria.

“Bioactive material”, “bioactive composition”, “biologically activematerial” or “bioactive formulation” refers to preparations, which arein such a form as to permit the biological activity of the bioactiveingredients to be unequivocally effective. Such bioactive materialsinclude but not limited to peptides, proteins, hormones, vitamins,minerals, drugs, microbiocides, fungicides, herbicides, insecticides,spermicides, nucleic acids, antibodies, and vaccines.

“Stabilizer or Stabilizing agent” refers to compounds or materials thatare added to the formulation to increase the viscosity of the wetformulation or to form a hydrogel. Examples of a suitable stabilizeragent include but are not limited to polysaccharides, such as, celluloseacetate phthalate (CAP), carboxy-methyl-cellulose, pectin, sodiumalginate, salts of alginic acid, hydroxyl propyl methyl cellulose(HPMC), methyl cellulose, carrageenan, guar gum, gum acacia, xanthangum, locust bean gum, chitosan and chitosan derivatives, collagen,polyglycolic acid, starches and modified starches, cyclodextrins andoligosaccharides (inulin, maltodextrins, raffinose, dextrans, etc.) andcombinations thereof.

“Protecting agent” or “protective agent” or “protectant” generallyrefers to compounds or materials that are added to ensure or increasethe stability of the bioactive material during the drying process andafterwards, or for long-term storage stability of the dry powderproduct. Suitable protectants are generally readily soluble in asolution and do not thicken or polymerize upon contact with water.Suitable protectants are described below and include, but are notlimited to, proteins such as human and bovine serum albumin, wheyprotein, soy protein, caseinate, gelatin, immunoglobulins, carbohydratesincluding monosaccharides (galactose, D-mannose, sorbose, etc.),disaccharides (lactose, trehalose, sucrose, etc.), an amino acid such asmonosodium glutamate, lysine, glycine, alanine, arginine or histidine,as well as hydrophobic amino acids (tryptophan, tyrosine, leucine,phenylalanine, etc.); a methylamine such as betaine; an excipient saltsuch as magnesium sulfate; a polyol such as trihydric or higher sugaralcohols (e.g., glycerin, erythritol, glycerol, arabitol, xylitol,sorbitol, and mannitol); propylene glycol; polyethylene glycol;Pluronics; surfactants, and combinations thereof.

A “stable” formulation or composition is one in which the bioactivemicroorganism or material therein essentially retains its viability,and/or biological activity upon storage. Stability can be measured at aselected temperature and humidity conditions for a selected time period.Trend analysis can be used to estimate an expected shelf life before amaterial has actually been in storage for that time period. For livebacteria, for example, stability is defined as the time it takes toloose 1 log of CFU/g dry formulation under predefined conditions oftemperature, humidity and time period.

“Viability” with regard to bacteria, refers to the ability to form acolony (CFU or Colony Forming Unit) on a nutrient media appropriate forthe growth of the bacteria. Viability, with regard to viruses, refers tothe ability to infect and reproduce in a suitable host cell, resultingin the formation of a plaque on a lawn of host cells.

The compositions and methods of the present invention solves the problemof providing a cost effective and industrially scalable drying processesfor producing a dry formulation containing bioactive microorganisms ormaterials, such as live or dead vaccines, bacteria, algae viruses and/orcell suspensions, peptides, proteins, hormones, vitamins, minerals,drugs, microbiocides, fungicides, herbicides, insecticides, spermicides,nucleic acids, antibodies, vaccines with a significantly extendedlifetime in the dry state. The invention provides a formulationcomprising a bioactive microorganism or material with a stabilizer agentand a protecting agent in a solution, cooling said formulation to atemperature above its freezing temperature, and stabilizing theformulation by removing the moisture under a regimen of reduced pressurewhile supplying heat to the composition.

Most of the viability loss of microorganism during drying processes canbe attributed to a combination of freeze-thaw stresses and ice crystalformation, high osmotic and oxidative stresses, shear forces and energyrelease during bubble cavitations associated with the “boiling” of thesolution under low drying pressure and high temperature. The presentinvention provides a formulation and an industrially scalable dryingprocess that minimizes losses during the drying and protects thebioactive microorganism under harsh storage conditions thereafter.

COMPOSITIONS OF THE INVENTION

The present invention includes formulation compositions of a bioactivemicroorganism or material, a stabilizer agent and a protecting agent ina viscous solution. The formulations of the invention were found to beinherently different in their physical structure and function fromnon-viscous or concentrated formulations that were dried withoutpre-cooling. For example, formulations of the prior art were initially“foamed” to facilitate effective drying. The foaming step generallyresulted in an extensive boiling and eruption of the solution that is anunavoidable consequence of the vacuum drying in a liquid state and as aresult, only a very low loading capacity of material in a vial or avessel can be achieved (see for example U.S. Pat. No. 6,534,087, inwhich the thickness of the final foamed product is less than 2 mm). Thecompositions and drying methods of the present invention allow only alimited and controlled expansion of the formulation thereby enablingmuch higher loading of material per drying area and, as a result, can beeasily scaled up to the production of large quantities of material.

Single cell microorganisms have been shown to benefit particularly fromthe formulations and drying methods of the present invention. In oneembodiment, the bioactive microorganism of the invention is probioticbacteria. The formulation is prepared according to the compositions andmethods of the invention including obtaining live probiotic bacteria inconcentrated solution, paste, frozen beads or dry powder from. Mixingthe probiotic bacteria under vacuum with a stabilizer agent and aprotecting agent, cooling the viscous formulation to a temperature aboveits freezing temperature, applying sufficient vacuum pressure tomaintain that pre-cooling temperature and supplying a heat source of 20°C. and above to facilitate water removal. Maintaining the pre-cooledtemperature of the formulation can be by conduction of heat away fromthe formulation, and/or by loss of latent heat due to water evaporation.To further accelerate the drying process a secondary drying step isapplied, at higher vacuum up to 0.1 Torr and at elevated temperature upto 70° C., to provide a final composition with water activity with an Awof 0.3 or less. Such a composition can remain stable in storageconditions of 40° C. and 33% RH for 60 days or more (see FIG. 1). Thespecified processes of the invention have shown to result in theunexpected ability of the cells to retain their viability beyond that ofestablished drying processes. The initial viability loss through theentire drying process according to the present invention was only 0.3logs (see FIG. 2).

Formulations for Preparation of Stable Dry Powder Compositions

The constituents to be mixed with the preferred microorganism ormaterial for the preparation of dry powder compositions according to theinvention, includes a stabilizer agent and protective agent. Suchconstituents, when mixed with the preferred bioactive microorganisms ormaterial, can be processed according to methods of the invention toprovide large quantities of stable dry compositions for storage andadministration of said microorganisms. The formulation stabilizers caninclude a mixture of a polysaccharide and an oligosaccharide. Thepreferred polysaccharide, particularly for stabilizing livemicroorganisms, was alginate. Because it was surprisingly found thatalginate is superior to other polysaccharides such as pectin and gumacacia in reducing the drying losses of sensitive biologicals such asprobiotics (FIG. 4). It was also preferred because of its hydrogelforming characteristics with non-toxic metals at mild temperatures.Alginate was also found to effectively stabilize the formulation undervacuum, by providing appropriate viscosity to the formulation andallowing a controlled expansion of the formulation at a particularviscosity (FIG. 3).

Combining an oligosaccharide with the alginate was also found to furthercontribute to the overall stability of the formulation. FIG. 5 shows theeffect on storage stability of different combinations of alginate andoligosaccharides. A combination of alginate and inulin was the preferredcombination in term of its long storage effect on the probioticbacteria. In one embodiment of the invention, at least one of theformulation stabilizer agents is preferably a gum that can form a firmhydrogel by cross-linking with metal ions.

Protective agents of the invention can include various proteins,peptides, sugars, sugar alcohols and amino acids. The protective agentis preferably one that does not crystallize and/or destabilize thebiologically active material in the formulation at freezing temperatures(e.g., −20° C.). It can be beneficial to include two or more differentprotective agents to inhibit the formation of crystals and stabilize thedried bioactive material formulation in storage conditions for long timeperiods.

The wet formulations can include a substantial amount of total solids(constituents minus the solvent, such as water). A major portion of thetotal solids can consist of the bioactive material, the stabilizer agentand the protective agent. For example, the bioactive material can bepresent in the formulation in a concentration ranging from about 2-50weight percent, the stabilizer agent from about 1-20 weight percent, andthe protective agent from about 20-80 weight percent. In anotherexample, the stabilizer agent can be present in the formulation in aconcentration ranging from about 0.5-10 weight percent, and theprotective agent from about 10-40 weight percent. Preferably, the wetformulation should have solids content between about 5% and 80%; morepreferably between about 30% to 60%. The viscosity of formulations ofthe invention are typically greater than 1000 centipoises (cP); morepreferably, greater than 10,000 cP and less than 450,000; and mostpreferably greater than 30,000 cP and less than 100,000 cP.

The viscosity of formulations of the invention can be as high as 450,000cP, provided that the protective agents are completely dissolved in thesolution. Highly viscous and homogenous slurries containing substantialamount of total solids can be achieved at elevated temperature,depending on the thermo and osmo-sensitivity of the bioactive material.For example, live cells formulations containing 30-60% of total solidscan be mixed at elevated temperature of about 35-40° C. and the mixingis carried out until all the protective agents are completely dissolved.

METHODS OF PREPARING STABLE DRY FORMULATIONS

Methods for preparing stable dry formulations for the preservation ofbioactive microorganisms include, obtaining a live culture of a specificmicroorganism in a concentrated solution, paste, frozen beads or drypowder from (stabilized or otherwise). Preparation of a formulation bymixing, under vacuum, the bioactive microorganism or material with astabilizer agent and a protecting agent in a solution, cooling theformulation to a temperature of no more than 10° C. above its freezingtemperature, and drying the formulation by evaporating the moistureunder reduced pressure while supplying heat to the formulation.

In one embodiment, for example, a formulation comprising a bioactivemicroorganism or material, a formulation stabilizer agent, and aprotecting agent are mixed to homogeneity, under mild vacuum of about10-50 Torr, in a solution. FIG. 6 shows the effect of differentdensities of the formulation on its expansion under vacuum. Theintroduction of air during mixing of the formulation constituents in asolution results in excessive and un-controllable foaming even atrelatively high vacuum pressure. The mixing under vacuum step accordingto the invention addresses this problem by eliminating the introductionof air or gas into the formulation solution, thereby eliminatingexcessive and uncontrolled foaming of the solution.

The solution is then cooled down to a temperature above its freezingpoint (usually between −5° C. and +5° C.). FIG. 7 shows the effect ofpre-cooling of the formulation solution on its expansion under vacuumpressure. It was surprisingly and unexpectedly found that boiling can beeffectively eliminated even under a relatively higher vacuum pressureand formulation expansion is better controlled when the solutiontemperature is reduced to no more than 10° C. above its freezingtemperature. As can be seen from FIG. 7, a vacuum pressure of 3 Torr canbe applied without excessive foaming provided that the formulation iscooled to +5° C. and preferably to −3° C.

Once cooled, the formulation is then dried under sufficient vacuum(e.g., about 3 Torr) to maintain that pre-cooled temperature during theprimary drying step. FIG. 8 shows the effect of the applied vacuumpressure on the temperature of the formulation solution. At relativelyhigh vacuum pressure above 8 Torr, the formulation temperature increasedto over 6° C. and will continued to rapidly increase toward the shelf orchamber temperature. At the same time, the solution will continuefoaming and further expanding. This embodiment is distinguished from theprior art discussed above (see for example U.S. Pat. No. 6,534,087,where the applied vacuum pressure is between 3-7 Torr and even higher),in which a stronger vacuum pressure is applied (<3 Torr) whilecontrolling the expansion of the formulation. This process results in asignificantly faster drying rate (see FIG. 9) and enables a high loadingcapacity of the formulation. In this embodiment, excessive foaming andboiling is eliminated even under much lower vacuum pressures because themethods of the invention provide a) a specific composition with acontrolled expansion under vacuum, b) a method that eliminates theintroduction of air into the formulation during mixing and c) asubstantial pre-cooling of the formulation.

Typical methods in the prior art involve extensive foaming and/orsplattering and violent boiling that can be damaging to sensitivebiologicals and cause difficulties for industrial scale up.Additionally, a complete and efficient degassing of viscous slurries isdifficult and may require an extended period of time. These obstacleswere resolved in the present invention by first carrying the entiremixing process under mild vacuum to eliminate the introduction ofentrained gasses into the formulation in the first place. Any smallamount of soluble gases that may remain in the formulation is thengently removed allowing the formulation to moderately expand under lowvacuum. The additional pre-cooling step of the formulation to atemperature above its freezing temperature provides an added control ofthe expansion rate and thereby allows much higher loading capacity perdrying area than was afforded according to the prior art. After theprimary drying stage is complete, the stabilized dry formulation can beheld at elevated secondary drying temperatures (up to 70° C.) and vacuumpressures of less than 0.2 Torr to complete drying of the formulation ina very short time.

Another embodiment of the invention provides methods to prepare hydrogelformulation compositions for preservation of bioactive microorganisms ormaterials. For example, a formulation containing a probiotic bacteria ina dry powder form, a stabilizer agent and a protective agent, are mixedin a solution, cross-linked to a hydrogel by adding metal ions ordivalent cations and then dried under low vacuum and temperature asdescribed above. The pre-cooled temperature of the formulation can bemaintained by conduction of heat away from the formulation, and/or byloss of latent heat due to water evaporation.

In one particular embodiment of the invention, for example, theformulation includes a concentrated fresh or frozen or dry culture oflive probiotic bacteria in a solution of 1 to 2.5% sodium alginate(preferably 1.5% sodium alginate), 1% to about 5% inulin (preferably2.5% inulin), 20% to 60% trehalose (preferably 40% trehalose) and 3% to15% casein hydrolysate (preferably 8% casein hydrolysate). Theformulation is mixed under vacuum at a temperature slightly above theroom temperature (typically between 25° C.-37° C.) until all thecomponents are completely dissolved.

In one additional embodiment of the invention, all the ingredients aredissolved in the solution at elevated temperature, then the slurry iscooled down to a temperature between 0° C. to −5° C. and a dry powder oflive microorganism is mixed in until all the components are completelydissolved. To facilitate the mixing of the dry live organism powder andto prevent clumping, a small amount of trehalose can be added to the drypowder (typically a mixture containing equal portions of dry powder andtrehalose is sufficient.

The formulation slurry is spread on trays at loading capacity of about200 g/sq ft and trays are placed on shelves in a freeze drier. The shelftemperature is adjusted to 0 to −5° C. (preferably −2° C.) and theslurry allowed to cool to that temperature. Vacuum pressure is thenapplied at 1 to 5 Torr (preferably 3 Torr) and shelf temperatureincreased to 20° to 45° C. (preferably 30° C.) for conductive heattransfer. The formulation temperature remained at about the temperature0 to −5° C. during the primary evaporation step to prevent the samplefrom freezing. Secondary drying step at maximum vacuum of 0.1 Torr andshelf temperature of 40° C. is started when product temperature reachedabout +10° C. The entire drying process proceeds for about 4 hours atwhich time the product is harvested and water activity is at Aw-0.3 orless.

In another embodiment of the invention, the loaded trays are pre-cooledto −2° C. in a cold room then immediately loaded in a vacuum oven drierfor radiant heat transfer. The primary and secondary drying steps arethen applied as described above for conductive heat transfer.

Preparing the Formulation

Formulations of the invention can include fresh, frozen or dry livemicroorganisms formulated into a solution or suspension containing aformulation stabilizer agent and a protective agent. The formulationstabilizer and/or high concentration of protective agent can bedissolved into a heated aqueous solution with agitation before coolingand mixing with the bioactive microorganisms. The microorganisms, suchas cultured virus or bacterium, can be concentrated and separated fromculture media by centrifugation or filtration, then directly mixed intothe formulation of the present invention, or added with conventionalcryoprotectants dropped into liquid nitrogen and the small frozen beadsstored at −80 C until mixed into the formulation. Alternatively, thefrozen beads can be freeze dried, milled into a fine powder, packed inair tight bags and stored refrigerated until mixed in the formulation ofthe invention. In one embodiment of the present invention, the totalityof the water in the formulation is provided in the liquid of theconcentrated live organism and the live organism suspension ismaintained at a temperature slightly above room temperature. The drycomponents of the formulation stabilizer agent and the protective agentare blended together and then slowly added to the warm suspension of thelive organism. The formulation suspension is gently agitated under mildvacuum in a planetary mixer until all components are fully dispersed anduniform slurry is obtained.

In another embodiment of the present invention the bioactivemicroorganism is in the dry powder form and is premixed dry withformulation ingredients before the resulting dry mixture is added towater at a temperature slightly above room temperature.

The bioactive microorganism or material can provide any bioactivity,such as enzymatic activity, induction of immune responses, cellularmultiplication, infection, inhibition of cell growth, stimulation ofcell growth, therapeutic effects, pharmacologic effects, antimicrobialeffects, and/or the like. The bioactive microorganism or material can benonliving cells or liposomes useful as vaccines or delivery vehicles fortherapeutic agents. Bioactive microorganism of the invention can be liveviruses and live attenuated viruses and/or the like.

Formulation stabilizers provide structural stability to the formulationand/or physical and chemical protective benefits to the bioactivemicroorganisms. The stabilizers can provide thickening viscosity to theformulation and better control over its expansion properties undervacuum pressure and increased structural strength to the driedformulation compositions of the invention.

The protective agents can include a variety of proteins, proteinhydrolysates, sugars, sugar alcohols and amino acids. For example,sugars such as sucrose or trehalose can physically surround thebioactive material to promote retention of molecular structurethroughout the drying process and impart structural rigidity to theamorphous matrix in the dry state. The protective agent can replacewater of hydration lost during drying, to prevent damage to cellmembranes and denaturation of enzymes. Other functions of the protectiveagents can include protecting the bioactive material from exposure todamaging light, oxygen, oxidative agents and moisture. Most protectiveagents can be readily dissolved in a solution in amounts ranging fromabout 0.1 weight percent to about 60 weight percent.

Pre-Cooling the Formulation

Formulations of the invention can be pre-cooled before applying vacuumpressure of the drying process, to provide benefits such as a furtherthickening of the formulation slurry, a better control over theexpansion of formulations under low vacuum pressure, stabilization ofbioactive microorganism or material, and/or enhancing the penetration offormulation constituents through cell membranes. Cooling can be appliedby any appropriate technique known in the art. For example, cooling canbe by contact and conduction with cold surfaces, loss of latent heat,and/or the like. Typically, formulations are held in vessels or spreadon metal trays and place in contact with a controlled temperaturesurface or a chamber where they equilibrate to the controlledtemperature. Typically, the formulations of the invention can bepre-cooled to a temperature above its freezing temperature (e.g.,between −5° C. and +5° C.).

Primary Drying of the Formulation

Typical processes for preservation of bioactive microorganisms such as,live or attenuated organisms include a combination of freezing andvacuum conditions that can result in membrane damage and denaturation ofcell constituents. The prior art teaches the use of higher vacuumpressures (e.g., less than 100 Torr), addition of specificcryoprotective agents, concentrating steps to obtain thick solutions(syrup), and/or higher initial temperatures to prevent freezing. The useof formulations and process parameters of the present invention overcomethese limitations and allow for higher loading capacity per drying areathat significantly improves industrial output.

The formulation in the present invention is dried by evaporation.Removal of solvent (moisture) from the gaseous environment around theformulation can be driven by condensation or desiccation. Evaporation ofsolvent from the formulation can provide accelerated primary drying ofthe formulation under low vacuum pressure. The controlled expansion ofthe formulation accelerates the primary drying of the formulation byrapid transfer of solvent out of the formulation. The controlledexpansion of the formulation is achieved by gentle degassing (notboiling) of the remaining dissolved gases when the drying vacuum isapplied. Since it is desirable not to boil or excessively foam theformulation because the cavitations and shear forces associated withbubble formation during boiling and/or the formulation may spill outfrom containment or have a negative impact on the bioactivemicroorganism.

As primary drying proceeds, the formulation structure is stabilized. Theheat supplied in the drying chamber compensates for the loss of latentheat caused by evaporation of solvent and the formulation temperature ismaintained within 10° C. above its freezing temperature. At some pointduring the primary drying process, the rate of evaporation of solventslows and the formulation temperature begins to increase due tosuperfluous supply of heat in the drying chamber. This point indicatesthe end of the primary drying step in this invention. As solvent isdriven out from the formulation, the protective agents in solutionbecome concentrated and thicker until it stops flowing as a liquid. Theamorphous and/or glassy formulation preserves a stable formulationstructure.

Secondary Drying

Secondary drying of the structurally stable formulation removes theremaining entrapped or bound moisture and provides a composition that isstable in storage for an extended period of time at ambienttemperatures. Secondary drying involves the application of elevatedtemperatures and a strong vacuum for several hours to days. In preferredembodiments the time period necessary to complete the secondary dryingstep is double the time of the primary drying step. Preferably, thewater activity of the formulation at the end of the secondary dryingstep is less than an Aw of 0.3. The drying temperature can range fromabout room temperature to about 70° C. A typical secondary dryingprocess for many bioactive microorganisms can include raising thetemperature from about 30° C. to about 40° C., and holding from about 30minutes to about 24 hours (preferably from about 30 minutes to about 4hours), to provide a stable dried formulation composition with wateractivity of less than an Aw of 0.3. In one particular embodiment of thesecondary drying, the drying temperature is slowly raised from primarydrying conditions at a rate that can further preserve the activity oflive biologicals such as live microorganisms. A strong vacuum can beprovided in the secondary drying process to accelerate the rate of waterremoval to lower residual moisture levels. The vacuum during thesecondary drying can be less than 1 Torr and, preferably, less thanabout 0.2 Torr.

The drying methods of the invention result in a biologically activemicroorganism or bioactive material that is encased within an amorphousglassy matrix, thereby preventing the unfolding of proteins andsignificantly slowing molecular interactions or cross-reactivity, due togreatly reduced mobility of the compound and other molecules within theamorphous glassy composition. As long as the amorphous solid is at atemperature below its glass transition temperature and the residualmoisture remains relatively low (i.e., below Aw of 0.3), the labilebioactive microorganism can remain relatively stable. It should be notedthat achieving a glassy state is not a prerequisite for long termstability as some bioactive microorganisms or materials may fare betterin a more crystalline state.

Preparation of Dry Powder

The dried formulation can be used en bloc, cut into desired shapes andsizes, or crushed and milled into a free flowing powder that provideseasy downstream processing like wet or dry agglomeration, granulation,tabletting, compaction, pelletization or any other kind of deliveryprocess. Processes for crushing, milling, grinding or pulverizing arewell known in the art. For example, a hammer mill, an impact mill, a jetmill, a pin mill, a Wiley mill, or similar milling device can be used.The preferred particle size is less than about 1000 μm and preferablyless than 500 μm.

The compositions and methods described herein preserve the biologicalactivity of the encased biologically active microorganism or bioactivematerials. For example, the compositions are tested for stability bysubjecting them at elevated temperature (e.g., 40° C.) and high humidity(e.g. 33% RH) and measuring the biological activity of the formulations.As an example for live probiotic bacteria, results of these studiesdemonstrate that the bacteria formulated in these formulations arestable for at least 60 days (see FIG. 1). Stability is defined as timefor one log CFU/g potency loss. Such formulations are stable even whenhigh concentrations of the biologically active material are used. Thus,these formulations are advantageous in that they may be shipped andstored at temperatures at or above room temperature for long periods oftime.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

Preparation of Dry Premixed Formulation:

Several formulation premixes were prepared according to Table 1.Trehalose was obtained from Cargill Minneapolis, Minn. Soy proteinisolate was obtained from Fearn Natural Foods, Mequon, Wis. Whey proteinConcentrate was obtained from Agri-Mark Inc., Middlebury, Vt. Caseinhydrolysate was obtained from Marcor, Carlstadt, N.J., and sodiumalginate from ISP Corp., Wayne, N.J. All ingredients were combinedtogether and uniformly mixed (Table 1).

TABLE 1 Formulations Premix composition (weight percent) Protein SoyWhey lydrolysate Constituent premix premix premix Sodium Alginate 3.03.0 3.0 Inulin 5.0 5.0 5.0 Trehalose 75.3 75.3 75.3 Soy protein Isolate14 — — Whey protein concentrate — 14 — Casein Hydrolysate 2.7 2.7 16.7

Example 2

Stable dry powder containing probiotic bacteria:

Lactobacillus Acidophilus (100 g frozen concentrate from a labfermentation harvest) was thawed at 37° C. Protein hydrolysate premix(100 g, Table 1) was slowly added to the thawed slurry of probioticbacteria in a jacketed dual planetary mixer (DPM, 1 qt, RossEngineering, Inc. Savannah, Ga.,). Mixing was carried out under mildvacuum (25 Torr) at 40 RPM and 37° C. for 10 minutes. The homogenousslurry was evenly spread on a tray at a loading capacity of 200 g/sq ftand the tray placed on a shelf in a freeze drier (Model 25 SRC, Virtis,Gardiner, N.Y.). Shelf temperature was set above the freezingtemperature of the slurry at −5° C. to cool, but not to freeze, theslurry. Vacuum pressure (3 Torr) was applied when the formulationtemperature reached about −1° C. The slurry starts to gently degas whenvacuum reached about 7 Torr. When the vacuum reached 3 Torr, the shelftemperature was increased to 50° C. The formulation temperature remainedat about −1° C. to about +5° C. during the first 50 minutes of primarydrying step. Once the formulation temperature increased to +10° C., thesecondary drying step was initiated. Maximum vacuum of 0.1 Torr wasapplied while still shelf temperature continued to maintain at 50° C.Secondary drying step was continued for additional 100 minutes, at whichpoint the drying process was terminated and the dry formula removed fromthe freeze drier. The water activity of the dry formulation at thispoint was Aw=0.23 as measured by a Hygropalm Awl instrument (RotonicInstrument Corp., Huntington, N.Y.).

The viability losses during formulation, preparation, and dryingprocesses are presented in FIG. 9. Viability losses during formulationpreparation were 0.26 logs and 0.34 logs during the drying process for atotal cumulative loss of 0.6 logs.

FIG. 10 shows the storage stability of the dry formulation underaccelerated storage conditions of 37° C. and 33% RH. After four weeks atthese storage conditions, the viability loss of the probiotic bacteriastabilized in the formulation of the invention was only 0.8 logs.

Example 3

Preparation of a Hydrogel Formulation:

Concentrated probiotic slurry was prepared according to Example 2 butusing the whey protein premix of Table 1. To this slurry, 0.5 g ofdibasic calcium phosphate was added, followed by 0.5 g ofgluconolactone. The slurry was allowed to harden at room temperatureover the next 2 hours to form a solid hydrogel. The firm gel was slicedto thin and long threads, using a commercially availableslicer/shredder. The thin threads were loaded on a tray at a loadingcapacity of 200 g/sq ft and placed in a freeze drier for drying asdescribed in Example 2. Four hours after establishing maximum vacuum of0.1 Torr, the dried product was taken out of the freeze drier. The wateractivity (Aw) of the formulation was 0.05 (Measured by HygroPalm Awl,Rotonic Huntington, N.Y.). The dry formulation was further ground tofine powder using standard hammer milling equipment and sieved through50-250 micron screens. FIG. 11 present a flow chart of the method ofproduction stable dry formulation from a hydrogel formulation accordingto the invention.

Example 4

Preparation of Probiotic Pet Food:

A commercially available pelleted pet food for dogs is dried in aconvection oven to a water activity of 0.1, and then coated with thestable probiotic dry formulation prepared as described in Example 3. Thedry pellets are sprayed with about 5% of fat-based moisture barrier (amixture of 40% chicken fat, 40% cocoa butter and 20% beeswax), mixed ina drum tumbler with the dry powder formulation (usually 0.1-0.5% oftotal pet food that provides a dosage of 10.sup.8 CFU/g), and finallysprayed with additional coat of the fat-based moisture barrier. Thetotal amount of coating is about 15% (of the pet food). Coating time isabout 30 min.

Example 5

Preparation of Fish Feed with Several Probiotic Microorganisms:

Pelleted feed for fish according to the present invention was preparedwith a mixture of several probiotics. A stable dry probiotic formulationcontaining a mixture of L, rhamnosus, L, acidophilus and Bifidobacteriumlactis was prepared as described in Example 2. A commercially availablestarter feed for salmon (Zeigler Bros., Gardners, Pa.) was first driedin a convection oven to a water activity of 0.1, and then coated withthe probiotics formulation in a drum tumbler. The pellets (100 g) werefirst sprayed with about 5% by weight of fat-based moisture barrier (amixture of 40% fish oil, 40% cocoa butter and 20% beeswax), then mixedwith 1 g of the stable dry probiotic formulation (to attain a dosage of10⁷ cfu/g feed), and finally sprayed with additional coat of thefat-based moisture barrier. The total amount of coating was about 10% ofthe fish feed.

Example 6

An Infant Formula Containing the Dry Formulation of the PresentInvention:

A stable dry formulation containing Lactobacillus GG (Valio Corp,Finland) is prepared according to Example 2 followed by a sieving intotwo particle size groups (above 50 μm and below 150 μm). An infantformula is prepared by mixing 99 g of an infant formula from MeadJohnson (Evansville, Ill.) with 0.1 g of the small size particles (below50 μm). The final product contains about 10⁸ cfu of Lactobacillus GG per100 g infant formula.

Example 7

Stable Dry Powder Containing an Enzyme:

A hydrogel formula containing 40 weight percent of proteases fromNovozymes (Denmark) is prepared by mixing, under mild vacuum, 60 g ofprotein hydrolysate formulation premix (Table 1) and 40 g of theproteases in 100 g of water solution. The wet formulation is dried in avacuum oven at a drying temperature of 50° C. For determination ofloading and storage stability of the dried formula: a dry sample isaccurately weighed (<100 mg) in a microcentrifuge tube. 200 μl ofdimethyl sulfoxide (DMSO) is added. The formulation is dissolved in theDMSO buffer by vortexing. To this sample, 0.8 ml of a solutioncontaining 0.05 N NaOH, 0.5% SDS and 0.075 M Citric acid (trisodiumsalt) is added. The tubes are sonicated for 10 min at 45° C., followedby a brief centrifugation at 5,000 rpm for 10 min. Aliquots of the clearDMSO/NaOH/SDS/Citrate solution are taken into wells of a microplate andanalyzed for protein content using the Bradford assay method. Thestorage stability of the stable enzyme formulation is significantlyhigher than a dry enzyme without the formulation of the presentinvention.

Example 8

Stable Dry Powder Containing Vitamin A:

A hydrogel formula containing 50 weight percent of Vitamin A (BASFCorp., Florham Park, N.J) is prepared by mixing, under 25 Torr vacuum,50 g of soy protein formulation premix (Table 1) and 50 g of vitamin Apowder in 100 g of water solution. The wet formulation is pre-cooled to−5° C., then spread on trays at a loading capacity of 200 g/sq ft anddried in a vacuum oven at an initial vacuum pressure of 3 Torr andtemperature of 70° C., followed by a maximum vacuum step of 0.2 Torr at70° C. once the formulation temperature reached to 5° C.

Example 9

Preparation of Invasive Species Bait:

Pelleted bait for specifically targeted invasive species according tothe present invention is prepared containing a pesticide. The wheyprotein premix of Table 1 is added to 200 gm of water. To this solutionis added 90 gm of rotenone and 0.5 gm of calcium phosphate dibasic,followed by 0.5 gm of gluconolactone. The slurry is allowed to harden atroom temperature over 2 hours. The firm gel is sliced to thin and longthreads through a slicer/shredder. The thin threads are loaded on a trayand placed in a vacuum oven dryer. Drying is stopped after achieving awater activity of 0.10. The dry formulation is ground to the appropriatesize distribution for the bait size specification for the specificspecies targeted.

Example 10

Preparation of a Protected Pesticide in a Water-Soluble Formulation:

A protected soluble granular formulation of a pesticide that wouldotherwise be subject to decomposition by other ingredients in aformulation during storage is prepared by the process of the presentinvention. The soy protein premix of Table 1 is added to 200 g of water.To this solution is added 80 g of a dry formulation of a sensitiveformulated pesticide. The slurry is transferred to a vacuum oven dryerand dried to a water activity of 0.1. The dry formulation is milled tothe desired size and packaged.

Example 11

Preparation of a Protected Pesticide in a Water Insoluble Formulation:

A protected insoluble granular formulation of a pesticide that wouldotherwise be subject to decomposition by other ingredients in aformulation during storage is prepared with the formulation and themethod of the present invention. The soy protein premix of Table 1 isadded to 200 g water. To this solution is added 90 g of a dryformulation of a sensitive pesticide and 0.5 g of calcium phosphatedibasic, followed by 0.55 g of gluconolactone. The slurry is allowed toharden at room temperature over 2 hours, and then sliced to thin, longthreads through a slicer/shredder. The thin threads are loaded on traysand dried in a vacuum oven dryer to reach a water activity of 0.1. Thedry formulation is further milled to the desired size distribution andpackaged.

Example 12

Ten (10) grams of dry Lactobacillus Rhamnosus GG is mixed with 100 g ofthe protein hydrolisate premix of Example 1 (table 1). This dry mixtureis slowly added to 100 gm of deionized water at 35° C. in a jacketeddual planetary mixer, and mixed for 10 minutes at 40 rpm. Thehomogeneous slurry is evenly spread on a tray at a loading capacity of100 gm/sq ft, and the tray is placed on a shelf in a freeze dryer (Model25 SRC, Virtis, Gardiner, N.Y.). The shelf temperature is set at 5° C.to cool the slurry. Vacuum is applied to reduce the pressure to 3 Torr,at which time the shelf temperature is raised to 30° C. After 2 hoursthe pressure is reduced further to 150 milliTorr with the shelftemperature still held at 30° C. Drying is continued for an additional 3hours at which point the product temperature has risen to within 2° C.of the shelf temperature. The dried product is then removed from thefreeze dryer and the water activity of the dry formulation at this pointis measured by a Hygropalm Awl instrument. Viability losses duringformulation, preparation and drying processes are measured and recorded.Storage stability testing of the dry formulation is conducted underaccelerated storage conditions of 32° C. and 20% RH.

Results for the trial at 30° C., and also for one repeated at 40 C areshown below

Water Activity after drying 0.25 0.26 Losses during drying 0.5 log 0.7log Losses during storage 0.4 log 0.7 log

Example 13

Twenty (20) grams of dry Lactobacillus Rhamnosus GG is mixed with 100 gwhey protein premix Example 1. This dry mixture is slowly added to 100gm of deionized water at 35° C. in a jacketed dual planetary mixer, andmixed for 10 minutes at 40 rpm. The homogeneous slurry is evenly spreadon a tray at a loading capacity of 100 gm/sq ft, and the tray is placedon a shelf in a freeze dryer (Model 25 SRC, Virtis, Gardiner, N.Y.). Theshelf temperature is set at 5° C. to cool the slurry. Vacuum is appliedto reduce the pressure to 3 Torr, at which time the shelf temperature israised to 30° C. After 2 hours the pressure is reduced further to 150milliTorr with the shelf temperature still held at 30° C. Drying iscontinued for an additional 3 hours at which point the producttemperature has risen to within 2° C. of the shelf temperature. Thedried product is then removed from the freeze dryer and the wateractivity of the dry formulation at this point is measured by a HygropalmAwl instrument. Viability losses during formulation, preparation anddrying processes are measured and recorded.

Storage stability testing of the dry formulation is conducted underaccelerated storage conditions of 32° C. and 20% RH.

Results for the trial at 30° C. and also for one repeated, but run at40° C. are shown below:

Water Activity after drying 0.23 0.26 Losses during drying 0.6 log 0.7log Losses during storage 0.8 log 0.7 log

Example 14

Ten (10) grams of dry Lactobacillus acidophilis are mixed with 10 gms oftrehalose and briefly set aside while 65.3 gm of trehalose, 3 gm ofsodium alginate, 5 gm of inulin and 16.7 gm of whey hydrolysate aremixed together as a dry powder and slowly added to 100 gm of deionizedwater at 35° C. in a jacketed dual planetary mixer, and mixed for 5minutes at 40 rpm. To this slurry is added the Lactobacillus acidophilisand trehalose dry premix, and the mixing is continued for an additional5 minutes at 35° C. The homogeneous slurry is evenly spread on a tray ata loading capacity of 100 gm/sq ft, and the tray is placed on a shelf ina freeze dryer (Model 25 SRC, Virtis, Gardiner, N.Y.). The shelftemperature is set at 5° C. to cool the slurry. Vacuum is applied toreduce the pressure to 3 Torr, at which time the shelf temperature israised to 30° C. After 2 hours the pressure is reduced further to 150milliTorr with the shelf temperature still held at 30° C. Drying iscontinued for an additional 3 hours at which point the producttemperature has risen to within 2° C. of the shelf temperature. Thedried product is removed from the freeze dryer and the water activity ofthe dry formulation at this point is measured using a Hygropalm Awlinstrument. Viability losses during formulation, preparation and dryingprocesses are measured and recorded. Storage stability testing of thedry formulation is conducted under accelerated storage conditions of 32°C. and 20% RH. Results for the trial where the dryer is maintained at30° C. and compared to those where the dryer is maintained at 50° C. areshown in Table below.

Water Activity after drying 0.23 0.26 Losses during drying 0.6 log 0.7log Losses during storage 0.8 log 0.9 log

Example 15

Ten (10) grams of dry Lactobacillus acidophilis are mixed with 10 gms oftrehalose and briefly set aside while 65.3 gm of trehalose, 3 gm ofsodium alginate, 5 gm of inulin and 16.7 gm of whey hydrolysate aremixed together as a dry powder and slowly added to 100 gm of deionizedwater at 50° C. in a jacketed dual planetary mixer, and mixed for 5minutes at 40 rpm. The slurry is cooled down to 4° C. To this cooledslurry is added the Lactobacillus acidophilis and trehalose premix, andthe mixing is continued for an additional 5 minutes at 4° C. Thehomogeneous slurry is evenly spread on a tray at a loading capacity of100 gm/sq ft, and the tray is placed on a shelf in a freeze dryer (Model25 SRC, Virtis, Gardiner, N.Y.). The shelf temperature is set at 5° C.to maintain the temperature of the cool slurry. Vacuum is applied toreduce the pressure to 3 Torr, at which time the shelf temperature israised to 30° C. After 2 hours the pressure is reduced further to 150milliTorr with the shelf temperature still held at 30° C. Drying iscontinued for an additional 3 hours at which point the producttemperature has risen to within 2° C. of the shelf temperature. Thedried product is removed from the freeze dryer. The water activity ofthe dry formulation at this point is Aw=0.23 as measured by a HygropalmAwl instrument. Viability losses during formulation, preparation anddrying processes total 0.6 logs.

Example 16

One hundred (100) gram of soy premix is slowly added to 100 gm ofdeionized water at 35° C. in a jacketed dual planetary mixer, and mixedfor 10 minutes at 40 rpm. Ten (10) grams of dry Bifidobacterium lactisBb-12 is added slowly with mixing at 20 rpm, and the slurry mixed for anadditional 5 minutes. The homogeneous slurry is evenly spread on a trayat a loading capacity of 100 gm/sq ft, and the tray is placed on a shelfin a freeze dryer (Model 25 SRC, Virtis, Gardiner, N.Y.).

The shelf temperature is set at 5° C. to cool the slurry. Vacuum isapplied to reduce the pressure to 3 Torr, at which time the shelftemperature is raised to 30° C. After 2 hours the pressure is reducedfurther to 150 milliTorr with the shelf temperature still held at 30° C.Drying is continued for an additional 3 hours at which point the producttemperature has risen to within 2° C. of the shelf temperature. Thedried product is removed from the freeze dryer and the water activity ofthe dry formulation at this point is Aw=0.26 as measured by a HygropalmAwl instrument. Viability losses during formulation, preparation anddrying processes total 0.7 logs.

Storage stability testing of the dry formulation under acceleratedstorage conditions of 32° C. and 20% RH show a viability loss of thestabilized probiotic bacteria in the formulation of the invention to beonly 0.7 logs after four weeks.

Example 17

The same parameters as Example #1, except the mixing done in the Rossmixer is under 25 inches of vacuum to give a slurry density of 1.2gm/cc. The water activity of the dry formulation at this point isAw=0.26 as measured by a Hygropalm Awl instrument. Viability lossesduring formulation, preparation and drying processes total 0.5 logs.

Storage stability testing of the dry formulation under acceleratedstorage conditions of 32° C. and 20% RH show a viability loss of thestabilized probiotic bacteria in the formulation of the invention to beonly 0.7 logs after four weeks.

Example 18

One hundred (100) grams of a fresh liquid concentrate of LGG bacteria(containing 10% solids and the rest water) is added to a jacketed dualplanetary mixer and warmed to 35° C. To this is slowly added 100 g ofwhey premix (Table 1). The resulting slurry is mixed for 10 minutes at40 rpm. The homogeneous slurry is evenly spread on a tray at a loadingcapacity of 100 gm/sq ft, and the tray is placed on a shelf in a freezedryer (Model 25 SRC, Virtis, Gardiner, N.Y.). The shelf temperature isset at 5° C. to cool the slurry. Vacuum is applied to reduce thepressure to 3 Torr, at which time the shelf temperature was raised to30° C. After 2 hours the pressure is reduced further to 150 milliTorrwith the shelf temperature still held at 30° C. Drying is continued foran additional 3 hours at which point the product temperature has risento within 2° C. of the shelf temperature. The dried product is removedfrom the freeze dryer. The water activity of the dry formulation at thispoint is Aw=0.25 as measured by a Hygropalm Awl instrument. Viabilitylosses during formulation, preparation and drying processes total 0.5logs. Storage stability testing of the dry formulation under acceleratedstorage conditions of 32° C. and 20% RH show a viability loss of thestabilized probiotic bacteria in the formulation of the invention to beonly 0.4 logs after four weeks.

Example 19

Lactobacillus Rhamnosus GG (LGG) One hundred (100) grams of unthawed,frozen concentrate and 100 g of protein hydrolysate premix were added toa jacketed dual planetary mixer (DPM, 1 pt, Ross Engineering, Inc.,Savannah, Ga.). This process can also be done by thawing the frozenconcentrate first. Mixing was carried out at 40 RPM and 37° C. for 10minutes. The homogeneous slurry was measured for viscosity (Brookfieldviscometer, Model #LVDVE115, Brookfield Engineering Laboratories, Inc.),and then evenly spread on a tray at a loading capacity of 100 g/sq ft.Viscosity Parameters for high viscosity ranges were: 300 g sample in a400 mL Pyrex beaker, 33-37 C, Spindle #64, 1.0 RPM speed, operatedwithout a guard-leg. The tray was then loaded into a −4° C. refrigeratorfor cooling for 30 min. After cooling, the drying began using a freezedrier (Model 25 SRC, Virtis, Gardiner, N.Y.) with a shelf temperatureset at 30° C. throughout, and 2800 mTorr of pressure for at least 2.5hours. After at least 2.5 hours, the pressure was decreased to 100 mTorrfor at least another 2.5 hours. This experiment was repeated with twodifferent batches of LGG fermentate, and included washing of one batchwith 3% DMV and reconstitution with de-ionized water prior to adding thehydrolysate premix.

CFU/g of final Losses during Viscosity of Sample product drying slurry(cP) LGG Batch # 1 2.20 × 10⁺¹⁰ 0.61 410,000 LGG Batch # 2 7.00 × 10⁺¹⁰0.48 N/A Washed LGG Batch # 2 1.38 × 10⁺¹¹ 0.21 319,000

Viscosity Parameters for medium viscosity ranges were: 300 g sample in a400 mL Pyrex beaker, 33-37° C., Spindle #64, 5.0 RPM speed, operatedwithout a guard-leg.

CFU/g of final Losses during Viscosity of Sample product drying slurry(cP) LGG Batch # 4 1.39 × 10⁺¹¹ 0.18 58,100 Washed LGG Batch # 4 1.31 ×10⁺¹¹ 0.23 36,200

Example 20

Lactobacillus Rhamnosus GG (LGG) One hundred (100) grams of frozenconcentrate was thawed at 37° C. and added to a jacketed dual planetarymixer (DPM, 1 pt, Ross Engineering, Inc., Savannah, Ga.). To it, 100 gof protein hydrolysate premix was added. Unthawed frozen concentrate mayalso be used. Mixing was carried out at 40 RPM and 37° C. for 10minutes, and then the slurry was evenly spread onto trays at a loadingcapacity of 100 g/sq ft. The trays were then loaded into a −4° C.refrigerator for cooling for 30 min. After cooling, the drying beginsusing a freeze drier (Model 25 SRC, Virtis, Gardiner, N.Y.) with a shelftemperature set at 30° C. throughout, and 2800 mTorr of pressure for atleast 2.5 hours. After at least 2.5 hours, the pressure was decreased to100 mTorr for at least another 2.5 hours. This same process was appliedto 10 g of dried (powdered) LGG material, which was mixed into 100 g ofprotein hydrolysate. This dry mixture was then slowly added to 90 g ofde-ionized water in the jacketed dual planetary mixer.

Sample Losses during drying (logs) Dry LGG/MM final product 1.26 FrozenLGG concentrate/MM final product 1.46

Example 21

Stable dry powder containing enzyme:

Forty (40) gram of proteolitic enzyme (Novozymes, Denmark) in the formof dry powder is mixed with 60 g of soy premix (Table 1). This drymixture is slowly added to 100 g of deionized water at 35° C. in ajacketed dual planetary mixer, and mixed for 10 minutes at 40 rpm. Thehomogeneous slurry is evenly spread on a tray at a loading capacity of100 gm/sq ft, and the tray placed on a shelf in a freeze dryer (Model 25SRC, Virtis, Gardiner, N.Y.). The shelf temperature is set at 5° C. tocool the slurry. Vacuum is applied to reduce the pressure to 3 Torr, atwhich time the shelf temperature is raised to 60° C. After 1 hour thepressure is reduced further to 150 milliTorr with the shelf temperaturestill held at 60° C. Drying is continued for an additional 1 hour atwhich point the product temperature had risen to within 2° C. of theshelf temperature. The dried product is removed from the freeze dryer.For determination of loading and storage stability of the dried formula:the dry sample is accurately weighed (<100 mg) in a microcentrifuge tubeand 200 μg of dimethyl sulfoxide (DMSO) is added. The formulation isdissolved in the DMSO buffer by vortexing. To this sample, 0.8 ml of asolution containing 0.05N NaOH, 0.5% SDS and 0.075M Citric acid(trisodium salt) is added. The tubes are sonicated for 10 min at 45° C.,followed by a brief centrifugation at 5,000 rpm for 10 min. Aliquots ofthe clear DMSO/NaOH/SDS/Citrate solution are taken into wells of amicroplate and analyzed for protein content using the Bradford assaymethod. The storage stability of the stable enzyme formulation issignificantly higher than a dry enzyme without the formulation of thepresent invention.

REFERENCES

The contents of the references cited herein are incorporated byreference herein for all purposes.

-   U.S. Pat. No. 6,964,771 Method for stably incorporating substances    within dry, foamed glass matrices. September 1997. Roser et al.-   U.S. Pat. No. 5,766,520 Preservation by formulation formation.    June 1998. Bronshtein-   U.S. Pat. No. 6,534,087 Process for preparing a pharmaceutical    composition. June 2001. Busson and Schroeder.-   U.S. Pat. No. 6,884,866 Bulk drying and the effects of inducing    bubble nucleation. April 2005. Bronshtein.-   U.S. Pat. No. 7,153,472 Preservation and formulation of living cells    for storage and delivery in hydrophobic carriers. December, 2006    Bronshtein-   20080229609 Preservation by Vaporization. June 2005. Bronshtein-   U.S. Pat. No. 6,306,345 Industrial scale barrier technology for    preservation of sensitive biological materials at ambient    temperatures. October 2001. Bronshtein et al.-   Morgan, C. A., Herman, N., White, P. A., Vesey, G. 2006.    Preservation of micro-organisms by drying; a review. J. Microbiol.    Methods. 66(2):183-93.-   Capela, P., Hay, T. K. C., & Shah, N. P. 2006. Effect of    cryoprotectants, prebiotics and microencapsulation on survival of    probiotic organisms in yoghurt and freeze-dried yoghurt. Food    Research International, 39(3) 203-211).-   Annear, 1962. The Preservation of Leptospires by Drying From the    Liquid State, J. Gen. Microbiol., 27:341-343.

What is claimed:
 1. A composition comprising (i) a bioactivemicroorganism or material as fresh, frozen or dry powder, (ii) at leasttwo stabilizer agents, and (iii) at least two protective agents, whereinthe composition is suitable for a liquid state drying and stabilizingthe bioactive microorganism or material.
 2. The composition of claim 1,wherein total solids range from about 30 weight percent to about 70weight percent.
 3. The composition of claim 1, wherein the bioactivemicroorganism or material is selected from a cell, a microbe, a virus, aculture, a probiotic, a yeast, an algae, a protein, a recombinantprotein, an enzyme, a peptide, a hormone, a vaccine, a drug, a vitamin,a mineral, a microbiocide, a fungicide, a herbicide, an insecticide or aspermicide.
 4. The composition according to claim 1, wherein thestabilizer agent is a polysaccharide and or an oligosaccharide.
 5. Thecomposition of claim 4, wherein the polysaccharides is selected fromcellulose acetate phthalate (CAP), carboxy-methyl-cellulose, pectin,sodium alginate, salts of alginic acid, hydroxyl propyl methyl cellulose(HPMC), methyl cellulose, carrageenan, guar gum, gum acacia, xanthangum, locust bean gum, chitosan and chitosan derivatives, collagen,polyglycolic acid, starches and modified starches, cyclodextrins andcombinations thereof.
 6. The composition of claim 4, wherein theoligosaccharide is selected from inulin, maltodextrins, dextrans andcombinations thereof.
 7. The composition of claim 4, wherein thestabilizers are present in an amount ranging from about 1 weight percentto about 20 weight percent.
 8. The composition according to claim 1,wherein the protective agents are readily soluble in a solution and donot thicken or polymerize upon contact with water.
 9. The compositionaccording to claim 1, wherein the protective agents are: proteins, suchas human and bovine serum albumin, egg albumen, gelatin,immunoglobulins, isolated soya protein, wheat protein, skim milk powder,caseinate, whey protein, pea protein and any protein hydrolysates;carbohydrates including, monosaccharides (galactose, D-mannose, sorbose,etc.), disaccharides (lactose, trehalose, sucrose, etc.), cyclodextrins;an amino acid such as lysine, monosodium glutamate, glycine, alanine,arginine or histidine, as well as hydrophobic amino acids (tryptophan,tyrosine, leucine, phenylalanine, etc.); a methylamine such as betaine;an excipient salt such as magnesium sulfate; a polyol such as trihydricor higher sugar alcohols, e.g. glycerin, erythritol, glycerol, arabitol,xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol;pluronics; surfactants; and combinations thereof.
 10. The composition ofclaim 9, wherein the protective agents are present in an amount rangingfrom about 1 weight percent to about 80 weight percent.
 11. Thecomposition of claim 1, wherein the total amount of the protectiveagents is between 20 and 70 weight percent.
 12. A method for preparing astable dry powder composition of claim 1, such method comprising: (i)combining under vacuum a bioactive microorganism or material in the formof fresh, frozen or dry powder, dry stabilizer agents and dry protectiveagents in an aqueous solvent; (ii) cooling the mixture of step (i) to atemperature above its freezing temperature; (iii) primary drying of thecooled mixture by evaporation, under vacuum, at a temperature above itsfreezing temperature; (iv) secondary drying of the mixture at atemperature of 20° C. or more for a time sufficient to reduce the wateractivity of the mixture to Aw −0.3 or less.
 13. The method of claim 12,wherein the mixture is cooled before drying to a temperature above itsfreezing temperature.
 14. The method of claim 12, wherein the drying ofthe mixture is done by evaporation.
 15. The method of claim 12, whereinthe temperature of the mixture during the primary drying step is aboveits freezing temperature.
 16. The method of claim 12, wherein thesecondary drying step is started when the temperature of the mixture isincreased by at least 10° C. above its initial drying temperature. 17.The method of claim 12, wherein the evaporation rate of the remainingsolvent during the secondary drying step is accelerated by increasingthe temperature and/or vacuum pressure.
 18. The method of claim 12,wherein the mixture is dried for a time sufficient to reduce theformulation to a water activity of Aw −0.3 or less.
 19. The method ofclaim 12, wherein the dried mixture is cut, crushed, milled orrespectively pulverized into a free flowing powder.
 20. The method ofclaim 19, wherein particle size of the dried mixture is less than about1000 μm.